Methods and apparatus for processing a sample of biomolecular analyte using a microfabricated device

ABSTRACT

A technique processes a sample of biomolecular analyte. The technique uses an apparatus having a support assembly that receives and supports a test module, a load assembly that loads the sample of biomolecular analyte onto the test module, an electrophoresis assembly that applies a current to the test module such that components within the sample separate by electrophoresis, and a controller that controls operations of the load assembly and the electrophoresis assembly. The load assembly and the electrophoresis assembly are coupled to the support assembly. The controller controls the operation of the load assembly in an automated manner. Preferably, the test module includes a dielectric plate member having an upper planar surface and a lower planar surface that is spaced apart from and coplanar with the upper planar surface. The dielectric plate member has at least one set of channels that includes an injection channel and a separation channel. The injection channel extends from the upper planar surface to the lower planar surface. The separation channel extends within the dielectric plate member in a plane parallel with the upper and lower planar surfaces and intersects the injection channel.

RELATED APPLICATIONS

[0001] This application is a divisional of application Ser. No.09/153,215, filed Sep. 14, 1998, which claims the benefit of U.S.Provisional Application No. 60/058,798, filed on Sep. 15, 1997.

[0002] The entire teachings of the above application(s) are incorporatedherein by reference.

GOVERNMENT SUPPORT

[0003] The invention was supported, in whole or in part, by Grant No.RO1 HG01389 from the National Institutes of Health and Grant No.F49620-95-1-0165 from the Defense Advanced Research Projects Agency/AirForce Office of Scientific Research. The Government has certain rightsin the invention.

BACKGROUND OF THE INVENTION

[0004] The human genome includes stretches of DNA composed of shorttandem repeats (STRs). The analysis of such STRs is an important toolfor genetic linkage studies, forensics, and new clinical diagnosticsbecause STRs are abundant and their locations have been mapped ingenomes.

[0005] A typical STR is less than 400 base pairs in length, and includesrepetitive units that are two to seven base pairs in length. STRs candefine alleles which are highly polymorphic due to large variationsbetween individuals in the number of repeats. For example, four loci inthe human genome CSF1PO, TPOX, THO1, and vWA (abbreviated CTTv) arecharacterized by an STR allele which differs in the number of repeats.Two repeating units are found at these loci: AATG for TPOX and THO1, andAGAT for CSF1PO and vWA.

[0006] In general, STR analysis involves generating an STR profile froma DNA sample, and comparing the generated STR profile with other STRprofiles. Generating an STR profile typically involves dying or taggingSTRs within a DNA sample, separating the tagged STRs within the sampleusing electrophoresis (applying an electric field), and recording thetagged STRs using a detector (e.g., a laser and a scanner).

[0007] One procedure for generating an STR profile uses an elongated gelplate (or slab gel) that is approximately 35 cm long. In general, thisprocess (hereinafter referred to as “the gel plate process”) involvesdepositing a tagged DNA sample on an area of the gel plate, separatingthe STRs within the tagged DNA sample on the gel plate usingelectrophoresis, and scanning the gel plate with a detector to recordthe tagged STRs. Typically, the gel plate process requires two to threehours to complete.

[0008] Another procedure for generating an STR profile uses a capillarythat is 50 to 75 microns in diameter. This process (hereinafter referredto as “the capillary process”) generally involves placing a tagged DNAsample at one end of a capillary, and drawing the sample through thecapillary using electrophoresis to separate the STRs. A detector recordsthe STRs by scanning a portion of the capillary.

[0009] Typically, STR separation is faster in the capillary process thanin the gel plate process. In general, an increase in electrophoresiscurrent results in an increase in STR separation speed, and a higherelectrophoresis current typically can be applied to the capillary thanto the gel plate because the capillary more easily dissipates heat(caused by the current) which would otherwise skew the separationresults. A typical capillary process requires between 10 minutes and onehour to complete.

[0010] Another procedure for generating an STR profile uses a microchip(or chip) made of durable transparent glass or plastic. A typicalmicrochip is a monolithic structure that is planar in shape. Such amicrochip includes multiple pairs of channels (channel pairs) that runin a coplanar manner with the plane of the microchip. An individual STRseparation can be performed at each channel pair. Each channel pairincludes a long channel and a short channel. The short channelintersects the long channel near one end of the long channel and at a 90degree angle. In some microchips, the short channel includes a jog whereit intersects the long channel such that portions of the short channelare parallel but not co-linear. Typically, a microchip is formed usingphotolithography and chemical etching techniques to produce channelstructures in fused silica wafers.

[0011] An STR separation process that uses a microchip (hereinafterreferred to as “the microchip process”) generally involves orienting themicrochip so that it and the channels within lie horizontally (i.e.,perpendicular to the direction of gravity) and depositing a sample oftagged DNA over a hole in the upper surface of the microchip thatconnects with one end of the short channel of a channel pair. Next, theDNA sample is drawn horizontally through the short channel usingelectrophoresis such that STRs within the sample are partially separatedalong the short channel. Then, a portion of the sample at theintersection of the long and short channels is further separated alongthe long channel using electrophoresis. A detector records the STRs in amanner similar to that of the gel plate and capillary processes.

[0012] The microchip process provides advantages over the gel plate andcapillary processes. First, the microchip process requires less time tocomplete than the gel plate and capillary processes because, in themicrochip process, very large STRs (which impede STR separation in thegel plate and capillary processes) are removed from the DNA sampleduring electrophoresis along the short channel and thus do not impedeSTR separation along the long channel. Accordingly, STR separation alongthe long channel takes no more than a few minutes. Second, a microchipmay include multiple channel pairs such that multiple samples can beseparated and scanned simultaneously. Nevertheless, significantdecreases in electrophoretic run-times would greatly increase the speedof STR analysis.

[0013] Conventional high-speed DNA genotyping using a microchip isdescribed in an article entitled “High-Speed DNA Genotyping UsingMicrofabricated Capillary Array Electrophoresis Chips”, AnalyticalChemistry, Vol. 69, No. 11, Jun. 1, 1997 on pages 2181 through 2186, theteachings of which are hereby incorporated by reference in theirentirety. Ultra-high-speed DNA sequencing using capillaryelectrophoresis chips is described in an article entitled“Ultra-High-Speed DNA Sequencing Using Capillary Electrophoresis Chips”,Analytical Chemistry, Vol. 67, No. 20, Oct, 15, 1995 on pages 3676through 3680, the teachings of which are hereby incorporated byreference in their entirety.

SUMMARY OF THE INVENTION

[0014] In general, the term “biomolecular analyte” refers to bothnon-synthetic and synthetic nucleic acids (e.g., DNA and RNA) andportions thereof, and biological proteins. Described herein arepractical ultra-fast techniques for allelic profiling of suchbiomolecular analyte. In particular, the techniques involve using amicrofluidic electrophoresis device to analyze short tandem repeats(STRs) within a DNA sample. An assay method of the present invention hasmade it possible to rapidly achieve baseline-resolved electrophoreticseparations of single-locus STR samples. In one embodiment, the assaypermits baseline-resolved electrophoretic separations of single-locusSTR samples in approximately 30 seconds. In addition, analysis ofsamples (e.g., PCR samples) containing loci defined or characterized byan STR which differs in the number or repeats is performed rapidly(e.g., at a rate which represents a 10-to-100-fold improvement in speedrelative to capillary or slab gel systems) using the allelic profilingassay described herein. For example, analyses of PCR samples containingthe four loci CSF1PO, TPOX, THO1 and vWA (abbreviated as CTTv) can beperformed in less than two minutes. This constitutes a 10-to-100-foldimprovement in speed relative to capillary or slab gel systems.

[0015] Also described herein is a separation device (or test module)useful in an allelic profiling assay of the present invention. Theseparation device includes a microfabricated channel device having achannel of sufficient dimensions in cross-section and length to permit asample to be analyzed rapidly. In one embodiment, the separation deviceconsists of a microfabricated channel 45 μm×100 μm in cross-section and26 mm in length, that is filled with a replaceable polyacrylamide matrixoperated under denaturing conditions at 50° C. A fluorescently labeledSTR ladder is used as an internal standard for allele identification.Samples analyzed by the assay method can be prepared by standardprocedures and only small volumes (e.g., 4 μL) are required peranalysis. The device is capable of repetitive operation and is suitablefor automated high-speed and high-throughput applications.

[0016] The term “test plate” is used hereinafter to refer to adielectric structure having an intersecting channel pair structure.Other terms that are interchangeable with the term test plate aremicrofabricated channel device, microchip, chip, electrophoresis chipand ME device.

[0017] An embodiment of the invention is directed to an apparatus forprocessing a sample of biomolecular analyte. The apparatus includes asupport assembly that receives and supports a test module, a loadassembly that loads the sample of biomolecular analyte on the testmodule, an electrophoresis assembly that applies a current to the testmodule such that components within the sample separate byelectrophoresis, and a controller that controls operations of the loadand electrophoresis assemblies. The load assembly and theelectrophoresis assembly are coupled to the support assembly. Thecontroller controls the operation of the load assembly in an automatedmanner.

[0018] The sample of biomolecular analyte may be disposed about a beadthat is magnetically attractable. To this end, the load assembly mayinclude an electromagnetic loading device that, in response to thecontroller, (i) electromagnetically carries the bead from a samplesource to the test module using electromagnetism, and (ii) releases thebead into the test module. The load assembly may further include anelectromagnetic unloading device that provides a force on the bead in adirection toward the test module and away from the electromagneticloading device in response to the controller. The bead can beconstructed such that it releases the sample of biomolecular analytewhen deposited in the test assembly.

[0019] Alternatively, the apparatus may include a capillary that, inresponse to the controller, (i) transfers the sample from a samplesource to the test module using electrokinetics, and (ii) terminatestransfer of the sample. The load assembly may further include a gasketthat forms a hermetic seal between the capillary and the test modulewhen the capillary transfers the sample from the sample source to thetest module. In this situation, it is unnecessary to contain thebiomolecular analyte in a magnetically attractable bead.

[0020] Preferably, the apparatus further includes a detection assembly,coupled to the support assembly, that detects the components within thesample. The detection assembly may include an actuating member, coupledto the support assembly, and a scanner, coupled to the actuating member.The scanner scans the test module. The actuating member moves thescanner, in response to the controller, between (i) a first positionadjacent the support assembly that receives and supports the test moduleand (ii) a second position adjacent the support assembly that receivesand supports another test module.

[0021] The apparatus may further include an injection assembly, coupledto the support assembly, that injects fluid into the test module tofacilitate separation of the components within the sample byelectrophoresis. Accordingly, the injection assembly can preload thetest module with a matrix in an automated manner prior to loading thetest module with the sample of biomolecular analyte.

[0022] The apparatus may further include a set of electrical connectionsthat provides power from a power supply to the test module. Accordingly,if the test module includes electrical circuitry (e.g., a heating devicefor providing heat to the sample), the test module can obtain power forthe circuitry from the apparatus.

[0023] The load assembly may further include a robotic device, coupledto the support assembly, having an arm and an actuator that moves thearm between a sample source and the test module. Additionally, the loadassembly may include a load device, coupled to the arm of the roboticdevice, that transfers the sample of biomolecular analyte from thesample source to the test module. Furthermore, the load assembly mayinclude a camera, coupled to the arm of the robotic device, thatdetermines a position of the arm of the robotic device and indicates theposition to the controller. The controller moves the arm of the roboticdevice according to the indicated position.

[0024] The support assembly of the apparatus may include a supportmember that supports multiple test modules. Furthermore, the apparatusmay include a detector that moves relative to the support member to scaneach of the multiple test modules supported by the support member.Alternatively, the support member may move the multiple test modulesrelative to the detector such that the detector scans each test module.

[0025] Another embodiment is directed to a method for loading a testplate with a sample of biomolecular analyte that is disposed about abead that is magnetically attractable. The method includes the steps ofactivating an electromagnetic loading device to attract and carry thebead, and positioning the electromagnetic loading device adjacent anopening of the test plate. The method further includes the step ofdeactivating the electromagnetic loading device to release the bead atthe opening of the test plate such that the sample of biomolecularanalyte is loaded on the test plate.

[0026] The method may further include the step of activating anelectromagnetic unloading device that provides a force on the bead in adirection away from the electromagnetic loading device and toward theopening of the test plate. Accordingly, any bead material remaining onthe electromagnetic loading device after the electromagnetic loadingdevice is deactivated will be attracted away from the electromagneticloading device and toward the test plate.

[0027] Preferably, the step of positioning the electromagnetic loadingdevice includes the step of actuating a robotic assembly that supportsthe electromagnetic loading device such that the electromagnetic loadingdevice moves to a programmed position relative to the test plate. Thisallows the sample to be positioned accurately and consistently from testto test.

[0028] Another embodiment of the invention is directed to a method forloading a test plate with a sample of biomolecular analyte that isdisposed within a solution. The method includes the steps of positioninga capillary adjacent an opening of the test plate, and activating anelectrokinetics device coupled to a capillary such that the sample isdrawn through the capillary onto the test plate by electrokinetics. Themethod further includes the step of deactivating the electrokineticsdevice such that the sample is no longer drawn through the capillary.The step of activating preferably includes the step of dispensing afluid droplet of the sample onto the test plate.

[0029] Another embodiment of the invention is directed to a test moduleassembly. The test module assembly includes a dielectric plate memberhaving an upper planar surface and a lower planar surface that is spacedapart from and coplanar with the upper planar surface. The dielectricplate member has at least one set of channels that includes an injectionchannel and a separation channel. The injection channel extends from theupper planar surface to the lower planar surface. The separation channelextends within the dielectric plate member in a plane parallel with theupper and lower planar surfaces and intersects the injection channel.Preferably, the injection channel intersects the separation channel at aright angle, and terminates at the upper and lower planar surfaces atright angles to the upper and lower planar surfaces. The plate member ispreferably wedge shaped.

[0030] The test module assembly may further include a housing thatattaches to the dielectric plate member. The housing provides (i) afirst opening over the upper planar surface such that a first end of theinjection channel is accessible through the first opening, and a secondopening over the lower planar surface such that a second (i.e.,opposite) end of the injection channel is accessible through the secondopening. The housing may further provide a third opening over either theupper planar surface or the lower planar surface such that theseparation channel is accessible to a detection device through the thirdopening.

[0031] Preferably, the test module assembly includes a heating device,supported by the housing, that provides heat to the dielectric platemember when power is provided to the heating device. The heatfacilitates the separation of components within the sample duringelectrophoresis.

[0032] The test module assembly may further include a first porousmembrane that covers the first end of the injection channel and a secondporous membrane that covers the second end of the injection channel toretain a matrix within the injection and separation channels. The firstand second porous membranes may be adhered to the dielectric platemember.

[0033] The test module assembly may further include an electromagneticunloading device, supported by the housing, that draws magnetic materialfor testing towards the dielectric plate member when the electromagneticunloading device is activated. Accordingly, a sample of biomolecularanalyte that is about a magnetically attractable bead will be drawn awaytoward the dielectric plate member when unloading the sample.

[0034] Preferably, the dielectric plate member includes multiple sets ofchannels. Each set of channels includes an injection channel and aseparation channel. The injection channel of each set extends from theupper planar surface to the lower planar surface. The separation channelof each set extends within the dielectric plate member in a planeparallel with the upper and lower planar surfaces and intersects theinjection channel of that set. The injection channels of the multiplesets of channels may be parallel with each other. These features of theinvention increase the channel density (i.e., the number of channel setsper unit area of the plate member) of the dielectric plate memberenabling more tests to be scanned by an individual scanner in a fixedposition relative to the dielectric plate member. Furthermore, theorthogonal orientation of the injection channels (relative to the planeof the plate member) improves initial separation of the componentswithin the sample during electrophoresis in the direction of theinjection channel.

[0035] Another embodiment of the invention is directed to a method forseparating a sample of biomolecular analyte. The method includes thestep of drawing the sample along a longitudinal axis of an injectionchannel of a test plate using electrokinetics. The test plate is planarin shape along an orthogonal axis to the longitudinal axis of theinjection channel. The method further includes the step of subsequentlydrawing the sample along the orthogonal axis through a separationchannel of the test plate using electrokinetics. The separation channelintersects the injection channel within the test plate.

[0036] The method may further include the steps of drawing multiplesamples along the longitudinal axes of multiple other injection channelsusing electrokinetics simultaneously with the step of drawing thesample, and drawing the multiple samples along other orthogonal axesthrough multiple other separation channels using electrokinetics. Themultiple other separation channels respectively intersect the multipleother injection channels.

[0037] Preferably, the method further includes the step of scanning arespective portion of the separation channel and the multiple otherseparation channels with a detection device.

[0038] Another embodiment of the invention is directed to a system foranalyzing short tandem repeats within a sample of biomolecular analyte.The system includes a test plate having a separation channel, a supportassembly that supports the test plate, an automated loading device thatloads the sample of biomolecular analyte on the test plate in anautomated manner, and an electrophoresis device that separates shorttandem repeats in the sample within the separation channel of the testplate. The automated loading device and the electrophoresis device arecoupled to the support assembly.

[0039] Preferably, the automated loading device includes a roboticactuator assembly, coupled to the support assembly, that obtains thesample from a sample source and deposits the sample at a particularlocation on the test plate.

[0040] Alternatively, the automated loading device includes a capillaryassembly, coupled to the support assembly, that obtains the sample froma sample source (e.g., using electrokinetics) and deposits the sample ata particular location on the test plate.

[0041] The system may further include a detection device that detectsthe short tandem repeats within the separation channel of the testplate.

[0042] Another embodiment of the invention is directed to method foranalyzing short tandem repeats within a sample of biomolecular analyte.The method includes the steps of providing a test plate having aseparation channel, activating an automated loading device that loadsthe sample of biomolecular analyte on the test plate in an automatedmanner, and connecting an electrophoresis device to the test plate andactivating the electrophoresis device to separate short tandem repeatsin the sample within the separation channel of the test plate.

[0043] Another embodiment of the invention is directed to a test moduleassembly that includes a rotatable dielectric plate member having anupper planar surface and a lower planar surface that is spaced apartfrom and coplanar with the upper planar surface. The rotatabledielectric plate member has multiple separation channels that extendwithin the dielectric plate member in a plane parallel with the upperand lower planar surfaces. The multiple separation channels extendradially from an inner portion of the dielectric plate member to anouter portion of the dielectric plate member.

[0044] Preferably, the rotary dielectric plate member is disk shaped andfurther includes multiple injection channels. Each injection channel mayintersect a corresponding separation channel within the outer portion ofthe dielectric plate member.

BRIEF DESCRIPTION OF THE DRAWINGS

[0045] The foregoing and other objects, features and advantages of theinvention will be apparent from the following more particulardescription of preferred embodiments of the invention, as illustrated inthe accompanying drawings in which like reference characters refer tothe same parts throughout the different views. The drawings are notnecessarily to scale, emphasis instead being placed upon illustratingthe principles of the invention.

[0046]FIG. 1 is a perspective view of a system for processingbiomolecular analyte according to the invention.

[0047]FIG. 2 is a side view of a load assembly and test module of thesystem of FIG.

[0048]FIG. 3 is a perspective view of an alternative load assembly forthe system of FIG. 1.

[0049]FIG. 4 is a perspective view of another system for processingbiomolecular analyte according to the invention.

[0050]FIG. 5 is a timing diagram of the operation of the system of FIG.2.

[0051]FIG. 6 is an exploded view of a test module that is usable by thesystems of FIGS. 1 and 4.

[0052]FIG. 7 is a side view of a test module when in use by one of thesystems of FIGS. 1 and 4.

[0053] FIGS. 8A-8C are top view portions of a microchip that is suitablefor use by the systems of FIGS. 1 and 4.

[0054]FIGS. 9A and 9B are views of a preferred microchip that issuitable for use by the systems of FIGS. 1 and 4.

[0055]FIG. 10 is a top view of the preferred microchip of FIGS. 9A and9B.

[0056]FIGS. 11A and 11B are perspective views of a scanning arrangementfor scanning a microchip that is usable by the systems of FIGS. 9A, 9Band 10.

[0057]FIG. 12A is a microchip electropherogram for the four loci CTTvallelic sizing standard.

[0058]FIG. 12B is a microchip electropherogram presenting the allelicprofile of an individual obtained by spiking a PCR amplified sample withthe CTTv sizing standard.

[0059]FIG. 13 is another microchip electropherogram for the four lociCTTv allelic sizing standard.

[0060]FIG. 14 is a graphic representation of predicted minimumseparation time required to achieve a resolution of 1.0 for the last twoalleles of the CSFLPO locus as a function of the injection plug width at200 and 500 V/cm in the presence of a 4% linear polyacrylamide (1×TBEbuffer with 3.5 M urea and 30% v/v formamide) sieving matrix at 50° C.

[0061]FIG. 15 is a graphic representation of predicted minimumseparation time required to achieve a resolution of 1.0 for the last twoalleles of each locus of the CTTv system as a function of the injectionplug width at 500 V/cm in the presence of a 4% linear polyacrylamide(1×TBE buffer with 3.5 M urea and 30% v/v formamide) sieving matrix at50° C.

DETAILED DESCRIPTION OF THE INVENTION

[0062] There is a compelling need for improved throughput and reducedcost for electrophoretic separation of biologically important molecules,especially for such applications as DNA sequencing and DNA typing.Traditional aspects of the supporting technology including traditionalpipette loading and labor-intensive preparation of gel plates haveconstrained attempts to miniaturize the apparatus and to increaseparallelism.

[0063] In contrast to conventional techniques, an embodiment of theinvention is directed to an automated technique for processing a sampleof biomolecular analyte with improved throughput and reduced preparationrequirements. The embodiment involves the use of an apparatus 20 forprocessing a sample 22 of biomolecular analyte, as shown in FIG. 1. Theapparatus 20 includes a support assembly 24, a load assembly 26, anelectrophoresis assembly 28, and a controller 30. The support assembly24 receives and supports a test module 32 including multiple channelpairs (channel sets for testing respective samples). The load assembly26 obtains one or more samples 22 from a sample source 34, and loads thesamples 22 simultaneously (in parallel) into respective channels/portson the test module 32. The electrophoresis assembly 28 applies a currentto the test module 32 such that components within the samples 22 (e.g.,STRs) separate by electrophoresis. The controller 30 controls theoperations of the load and electrophoresis assemblies 26,28.

[0064] The load assembly 26 includes a robotic device 36 having arobotic arm 38 and an actuation assembly 40. The load assembly 26further includes a fluid transfer device 42 that is attached to therobotic arm 38. The fluid transfer device 42 has an array ofelectrically conductive fluid (sample) dispensing tips that carry samplematerial from the sample source 34 to respective injection channels inthe test module 32. The actuation assembly 40 moves the robotic arm 38and the fluid transfer device 42 according to a robot signal provided bythe controller 30. In particular, the actuation assembly 40 includesactuators 40X, 40Y and 40Z that are capable of moving the robotic arm 38and the fluid transfer device 42 along the X-axis, Y-axis and Z-axis,respectively. Preferably, the robotic device 36 provides both linearmotion (along any combination of the X, Y and Z axes) and rotary motion(about the axes).

[0065] During operation, the fluid transfer device 42 interfaces withboth the sample source 34 (e.g., a microtitre plate) and the test module32 (e.g., a microchip assembly). Preferably, each dispensing tip in thearray of dispensing tips of the fluid transfer device 42 includes anelectromagnetic loading device 50 having a core 52 and windings 54, asshown in FIG. 2. The controller 30 activates the electromagnetic device50 by providing a carry signal to the windings 54. The sample 22 ofbiomolecular analyte preferably is disposed about magneticallyattractable beads 56. The controller 30 positions the fluid transferdevice 50 over the sample source 34 and activates the electromagneticdevice 50. In turn the core 52 becomes magnetically charged such that adistal or working end 58 of the core 52 attracts, picks up and retainsthe beads 56. Then, the controller 30 operates the robotic device 36 tomove the fluid transfer device 42 over the test module 32 such that thedistal end 58 of the core 52 is adjacent a target opening 60 (injectionport) of the test module 32, as shown in FIG. 2. Next, the controller 30discontinues providing the carry signal to deactivate theelectromagnetic loading device 50. Accordingly, the core 52 becomesdemagnetized and the beads 56 are released from the end 58 of the core52 and drop into the target opening 60 of the test module 32.

[0066] As will be discussed later in further detail, the test module 32includes a microchip that contains an internal sieving matrix and amicrofabricated coverplate. The test module 32 may be driven with a highvoltage supply 43 and a temperature controller 45 (see FIG. 1).

[0067] The load assembly 26 may further include an electromagneticunloading device 62, as shown in FIG. 2. The electromagnetic unloadingdevice 62 is positioned beneath the target opening 60 such that thetarget opening 60 of the test module 32 is between the electromagneticloading device 50 and the electromagnetic unloading device 62. When thecontroller 30 discontinues providing the carry signal to deactivate theelectromagnetic loading device 50, the controller 30 simultaneouslyprovides a release signal to the electromagnetic unloading device 62 todraw the beads away from the end 58 of the core 52 and toward the targetopening 60 of the test module 32. Accordingly, the electromagneticunloading device 62 provides a magnetic force on any beads that may haveotherwise stuck to the end 58 of the core 52 to unload them into thetarget opening 60.

[0068] As an alternative to the robotic device 36 (see FIG. 1) and theelectromagnetic devices 50,62 (see FIG. 2), the load assembly 26 mayinclude a capillary assembly 70, as shown in FIG. 3. The capillaryassembly 70 includes multiple capillaries 72 (one shown), a rigidsupport 74 and a non-rigid gasket 76. When the rigid support 74 ispositioned properly over the test module 32, the gasket 76 forms ahermetic, liquid-tight seal with the test module 32 to preventcontamination. The rigid support 74 positions the proximal ends of thecapillaries 72 such that they match with corresponding target openings60 of the test module 32. In particular, the rigid support 74 providesappropriately sized and spaced holes and structural support to maintainthe proximal ends of the capillaries rigidly in place. Preferably, thecapillaries are made of glass and the proximal ends of the capillariesextend through the rigid support 74 and at least a portion of thenon-rigid gasket 76. The other (opposite) ends of the capillaries 72mate with the sample source 34 in a conventional manner. In particular,the other ends of the capillaries 72 connect with wells of a microtitreplate of the sample source 34.

[0069] For the capillary assembly of FIG. 3, the sample source 34includes samples of biomolecular analyte in fluid form. The capillaryassembly 70 injects the samples into the test module 32 by transferringthe samples from the source to the test module 32 electrokinetically.That is, the load assembly 26 provides a voltage between the samplesource 34 (e.g., microtitre plate wells) and the test module 32 (e.g.,microfluidic channels within a microchip) such that the samples migratefrom the sample source 34 to the test module 32. Preferably, eachcapillary dispenses a sample fluid droplet onto the test module 32. Sucha droplet may be released by a physical pulse (e.g., from a pulsedevice). An advantage of such a system is the ability to load multiplesamples simultaneously through multiple capillaries onto the test module32 and into multiple injection channels therein.

[0070] Reference is made back to FIG. 1 for a further discussion of theapparatus 20. After the test module 32 has been loaded with one or moresamples 22 from the sample source 34, the electrophoresis assembly 28applies an electrical current to the test module 32 to separate thecomponents (e.g., STRs) within each sample 22. The apparatus 20preferably includes a detection assembly 80 which then detects theseseparated components. The detection assembly 80 includes a scanner 82and an actuator 84 that moves the scanner 82 to various scanningpositions relative to the support assembly 24. In particular, theactuator 84 moves the scanner 82 to a pertinent portion of each channelset of the test module 32 to detect respective separation components.The detection assembly 80 collects data from the test module 32 andprovides the data electronically to a data storage system of thecontroller 30.

[0071] Preferably, the support assembly 24 is capable of receiving andsupporting multiple test modules 32, as shown in FIG. 1. In thissituation, the actuator 84 of the detection assembly preferably movesthe scanner 82 between multiple positions over the multiple test modules32 (e.g., between a first position adjacent a first test module and asecond position over a second test module as well as multiple locationswithin each of the first and second positions for detecting respectivemultiple channels and the separated components therein).

[0072] Preferably, the apparatus 20 further includes an injectionassembly 86 that injects fluid (e.g., a separation matrix) into eachtest module 32 prior to sample loading. The fluid facilitates separationof the sample components during electrophoresis. Such automated loadingenables injection of ultrathin gels (eliminating manual pouring of gels)and permits the use of unbonded microchips (greatly reducing the costand increasing the re-usability of the microchips).

[0073] The apparatus 20 may further include power supply connections 88that provide power to each test module 32 from the voltage supply 43.Accordingly, circuitry within each test module 32 (e.g., a heater) canbe powered to facilitate component separation during electrophoresis.

[0074] It should be understood that the apparatus 20 provides sampleloading, sample separation and sample detection of one or more samplesin parallel, in an automated manner. That is, such operations can beperformed without human intervention. As such, the robotic loading ofthe sample 22 is easily repeatable and requires minimal preparation. Inparticular, the controller 30 stores programmed positions enabling thefluid transfer device 42 of the load assembly 26 to automaticallytransfer samples 22 from the sample source 34 to respectiveports/channels of each test module 32 for testing. Accordingly,labor-intensive sample loading, which is common with conventionaltechniques, is unnecessary when using the apparatus 20.

[0075] Another apparatus 90 for processing a sample of biomolecularanalyte is shown in FIG. 4. The apparatus 90 is similar to the apparatus20 of FIG. 1. In particular, the apparatus 90 includes a supportassembly 92, a load assembly 94, an electrophoresis assembly 96 and acontroller 110 that operate in manners similar to those of the apparatus20 as will now be explained.

[0076] The load assembly 94 loads samples onto test modules 32 that arereceived and supported by the support assembly 92. The load assembly 94includes a fluid transfer device 100 and a robotic device 102 that movesthe fluid transfer device 100 in a manner similar to that of the roboticdevice 36 of FIG. 1. The fluid transfer device 100 (e.g., a multi-tippipetter) is capable of transferring multiple sample sources 104, inparallel/simultaneously, to one or more test modules 32. Additionally,the fluid transfer device 100 may operate as an injection device topreload the test modules 32 with fluid prior to sample loading.Accordingly, there is no need for a separate injection assembly (asshown in FIG. 1).

[0077] The apparatus 90 further includes a wash station 98 that washesthe fluid transfer device 100 between sample transfers to avoidcontamination between the samples. The wash station 98 preferably washesthe fluid transfer device 100 before and after each of the fluidinjection and sample loading procedures.

[0078] The apparatus 90 may further include other features of theapparatus 20 of FIG. 1. In particular, the apparatus 90 may include adetection assembly 106 which resides below the test modules 32. In amanner similar to the detection assembly 80 of FIG. 1, the detectionassembly 106 of FIG. 4 moves relative to the test modules 32 and thesupport assembly 92 to selectively scan the test modules 32 (i.e.,multiplex between the test modules 32) after electrophoresis.

[0079] Preferably, the load assembly 94 of the apparatus 90 includes acamera 108 that sends an alignment signal to the controller 110. Thealignment signal indicates the position of the camera 108 and the fluidtransfer device 100 relative to positions on the support assembly 92(e.g., positions over the sample sources 104 and the test modules 32).The controller 110 moves the fluid transfer device 100 roboticallybetween the sample sources 104, the wash station 98 and the test modules32 according to the alignment signal. In particular, the controller 110provides a robot signal to the robotic device 102 to control themovement of actuators of the robotic device 102.

[0080] Within the test module 32 is a microchip (also called an “MEdevice”). It should be understood that the automated features of theapparatus 90 of FIG. 4 (as well as the apparatus of FIG. 1) provide abridge between the conventional macroscopic format (millimetergeometries) of microtitre plates and the microscopic (micrometergeometry) format of ME devices. That is, the current invention solvesthis problem by close integration of a precision motion control systemwith the ME device. Both the motion control system and the ME device arespecifically customized with microfabricated structures to achieve anefficient overall solution to the required format change.

[0081] Further details of the fluid transfer device 100 will now bediscussed. The apparatus 90 of FIG. 4 accurately translates a precisionmanufactured fluidic transfer head of the fluid transfer device 100which transfers microliter fluid quantities. An optical system (thecamera 108) is mounted on the load assembly 94, and computer hardwareand driver software of the controller 110 control the motion of thefluid transfer head allowing it to perform pre-programmed, automatedrepetitive procedures. Affixed to an active arm of this fluid transferhead is a multi-tip array of specialized fluid-handling tools 110capable of collecting and dispensing microliter amounts of fluidaccurately and reproducibly. The robotics and associated optics serve toalign and position the tip array over an industry-standard multiple-wellmicrotitre plate and collect the desired amount of liquid sample. Themotion system then lifts the tip array to the ME device of the testmodule 32, aligns to the ME device using the robotic optics systemdescribed above, and instructs high-precision pumps to dispense thesamples from the tip array into the appropriate microfabricatedinjection ports (each approximately 100 μm in diameter) on the ME deviceof the test module 32. This cycle is repeated until all of the injectionports on the ME device of the test module 32 have been loaded.Accordingly, the automated features of the apparatus 90 solve the formattransformation problem by bridging the conventional macroscopic format(millimeter geometries) of microtitre plates with the microscopic(micrometer geometry) format of microchips of the test modules 32.

[0082] Once the ME device is loaded, the electrophoresis assembly 96separates the samples based on their molecular weight and mobilitythrough a microchip sieving medium. In one embodiment, an opticallaser-induced fluorescence system is used to excite fluorescently taggedmolecules. The resultant signal is collected by a series of optics andphotomultiplier tubes or charge-coupled device cameras in the detectionassembly 106 and analyzed using software appropriate for the separationbeing done.

[0083] It should be understood that the apparatus 20 of FIG. 1 and theapparatus 90 of FIG. 4 support the use of multiple test modules 32 (eachtest module 32 being capable of simultaneously testing multiple samplesitself). In particular, the apparatus 20,90 can perform electrophoresison one test module 32 while preparing another. FIG. 5 shows a timingdiagram for two test modules 32 used by the apparatus 20 of FIG. 1 andby the apparatus 90 of FIG. 4. As shown, while a first test module 32undergoes injection and electrophoresis, another test module undergoesother procedures such as sample removal, cleaning and sample loading. Asa result, the various assemblies of the apparatus 20,90 are multiplexedbetween the multiple test modules 32.

[0084] Further details of the test module will now be provided withreference to FIG. 6 which shows an exploded view of a test module 32.The test module 32 includes a microchip 120, a lower housing member 122and an upper housing member 124. The lower and upper housing members122,124 form a housing that protects and supports the microchip 120. Themicrochip 120 is planar in shape with a top surface (an upper planarsurface) and a bottom surface (a lower planar surface) spaced apart fromthe top surface. The microchip is preferably wedge (or triangle) shaped.One end of the top surface has a set of injection ports 126 that operateas target openings that receive injection fluid, test samples andelectrophoresis electrodes. The microchip 120 includes channels 128 thatextend within the microchip 120 along a plane with the planes of themicrochip 120 surfaces. The top surface of the microchip 120 furtherincludes additional openings 130 that allow air to escape when materialis injected into the channels 128 and to provide access for additionalelectrophoresis electrodes.

[0085] The upper housing member 124 has a set of openings 132 that matchthe target openings 126 of the microchip 120. The openings 132 provideaccess to the target openings 126 when the upper housing member 124covers the microchip 120. In a similar manner, the upper housing member124 has other openings 134 and 138 to provide access (e.g., light andelectrophoresis electrodes) to the microchip 120 when the microchip ishoused by the housing members 122,124.

[0086] The upper housing member 124 further includes a slot 136 and thelower housing member includes a corresponding slot 140. The slots 136,140 allow light to pass from one slot to the other through the microchip120 when the microchip 120 is housed by the housing members 122,124.This allows a detection assembly to scan particular areas of thechannels 128 within the microchip 120 after (or during) electrophoresis.In particular, the matching slots 136,140 provide optical access forlaser-induced fluorescence detection.

[0087] The lower housing member 122 further includes a heating element142 (a heater) that provides heat to the microchip 120 when power isprovided to the heater through electrical connections 144 (e.g.,high-voltage connections) of the heating element 142. The heatfacilitates STR separation within the channels 128. Preferably, theheating element 142 is a ceramic device with high thermal conductivities(above 10 Watts/(meter Kelvin)) and high dielectric strengths (above 50volts per mil). Alumina, beryllia, and boron nitride ceramics aresuitable for the ceramic device.

[0088]FIG. 7 provides a schematic view of a test module 32 (FIG. 6)disposed on the apparatus 90 of FIG. 4. Electrophoresis electrodes areconnected at the openings 126,130 of a channel 128 of the microchip 120.The electrodes lead to a power supply through voltage relays that areswitchably controlled by the controller 110 (see FIG. 4). The controller110 further provides temperature control for the heating element 142(heater block). Additionally, the detection assembly 106 (scanner andlaser) scans a portion of the channel 128 of the microchip 120 to recordSTR separations.

[0089] Further details of the microchip 120 of the test module 32 willnow be provided with reference to FIGS. 8A, 8B and 8C, which providevarious partial top views of the microchip 120. The microchip 120includes multiple sets of channels 128, one set being shown in each ofFIGS. 8A-8C. Each set of channels (or channel set) includes an injectionchannel 150 and a separation channel 152. The injection and separationchannels 150,152 of each set 128 are coplanar with the plane of themicrochip 120. The separation channel 152 is longer than the injectionchannel 150 and intersects the injection channel 150 at a right angle(90 degrees). The injection channel 150 has a jog where it intersectsthe separation channel 152 to allow precise control of the amount ofsample to be separated within the separation channel 152.

[0090] Further explanation of the electrophoresis operation will now beprovided. First, a load assembly injects a sample into the injectionchannel 150 at the negative electrode end, as shown in FIG. 8A. Second,an electrophoresis assembly performs electrophoresis along the injectionchannel 150 causing the sample to partially migrate and separate in thedirection of the positive electrode. A portion of the sample becomespositioned within the jog 300 during electrophoresis along the injectionchannel 150. Next, as shown in FIG. 8B, the electrophoresis assemblyperforms electrophoresis along the separation channel 152. The portionof the sample within the jog 300 travels down the separation channel 152and separates further. Other portions of the sample remain in theinjection channel 150. The particular amount of sample separated in theseparation channel 152 can be precisely controlled by the diameter ofthe separation channel and the length of the jog 300. Then, as shown inFIG. 8C, the portion of the sample separates into components along theseparation channel 152 as electrophoresis continues along the separationchannel 152. In particular, movement of the components within theseparation channel 152 is from the negative electrophoresis electrode(at one end of channel 152) to the positive electrophoresis electrode(at the opposite end of the separation channel 152). Since very largecomponents of the sample remain in the injection channel 150, componentseparation within the separation channel 152 is less hindered. Finally,the separated components can be detected near the end of the separationchannel 152 having the positive electrode.

[0091] In one version of the microchip 120, the injection channel 150 is10 mm in length and the separation channel 152 is 35 mm in length. Theseparation channel 152 divides the injection channel 150 into two 5 mmportions, and the injection channel 150 divides the separation channel152 into a 5 mm portion and a 30 mm portion.

[0092] To create more compact structures in the microchip 120, thegeometry of the separation channel 152 can be extended in length andfolded. For example, the separation channel 152 can have a foldedchannel that is approximately 100 mm in length, or even approximately300 mm in length.

[0093] Conventionally, the injection channel 150 extends along a planethat is coplanar with that of the microchip 120 surfaces in the samemanner as the separation channel 152. Accordingly, when the microchip120 is disposed horizontally during electrophoresis, the injectionchannels 150 and the separation channels 152 are perpendicular with thedirection of gravity. In this arrangement, all of the openings in themicrochip are on the top surface of the microchip 120.

[0094] In a preferred microchip embodiment, the separation channelsextend along a plane that is coplanar with the surfaces of the microchipin a manner similar to that of conventional separation channels.However, in the preferred embodiment, the injection channels extend fromthe top surface to the bottom surface of the microchip and areorthogonal to the plane of the microchip surfaces, as shown in FIGS. 9Aand 9B. That is, the opening of the injection channel 150 that receivesthe sample and provides access to an electrophoresis electrode 160(i.e., the injection port) is at the upper surface, and the otheropening that provides access to another electrophoresis electrode 161 isat the bottom surface. When the microchip 120 is oriented such that itsplane is horizontal (i.e., perpendicular to the direction of gravity),the injection channel 150 of the preferred embodiment is oriented in thevertical direction (i.e., with the direction of gravity). Preferably,the injection channel is at right angles with the top and bottomsurfaces of the microchip 120.

[0095] The microchip 120 may include a well-shaped indentation 164around the injection port opening, as shown in FIG. 9A. The indentationhelps hold the sample over the receiving port of injection channel 150when the sample is introduced to the microchip 120. Preferably, porousmembranes 166,168 cover the openings of the injection channel 150 tohold the injection fluid (i.e., a separation matrix) within theinjection channel 150. The membranes 166,168 can be adhered to themicrochip 120. The pores of the membranes 166,168 should be small enoughto prevent the injection fluid from passing through. Suitable membranesinclude polyvinylidene fluoride membrane filters (either hydrophylic orhydrophobic types) with pore sizes of 0.1 micron to 0.65 micron, andpolytetrafluoroethylene membrane filters of 0.2 to 5.0 micron poresizes. Such filters are available from Millipore Inc. of Bedford, Mass.Also shown in FIG. 9A are the electrophoresis electrodes 170,172 for theseparation channel 152.

[0096]FIG. 9B shows top and bottom views of a preferred geometrymicrochip 120 having three sets of channels. The top view shows theindentations 164 in the top surface of the microchip 120 for each set ofchannels. Although the separation channels 152 are shown extending alongthe microchip 120, the injection channels 150 are shown on end in boththe top and bottom views.

[0097] It should be understood that the vertical orientation of theinjection channels 150 permits the channel sets to be densely clusteredon a simple microchip 120. In particular, more channel sets withvertical injection channels are able to be clustered in a microchip areathan channel sets with non-vertical injection channels. Accordingly,more samples can be separated in a particular microchip area, and moreseparation channels can be scanned by a detection assembly withouthaving to substantially move the detection assembly relative to themicrochip.

[0098] Furthermore, it should be understood that the vertical injectionchannel of the microchip of FIGS. 9A and 9B may have a geometry similarto the horizontal injection channel FIGS. 8A-8C. That is, the verticalinjection channel may include a jog where it intersects the separationchannel such that a cross-section of the vertical injection channelmicrochip looks similar to FIGS. 8A-8C.

[0099]FIG. 10 shows a preferred arrangement of channel sets on awedge-shaped microchip 120. The injection channels 150 are spaced apartto provide enough room for samples to be injected into the injectionports without risk of sample cross-mixing between injection ports. Suchspacing is on the order of millimeters which is less suitable for humanload handling than the automated loading means discussed above in FIGS.1 and 4. The separation channels 152 are allowed to be spaced moreclosely together to enable simultaneous scanning at the narrow end ofthe wedge shape.

[0100] As stated above in connection with the apparatus 20 of FIG. 1 andthe apparatus 90 of FIG. 4, the detection assembly may move between testmodules 32 to multiplex the use of a single scanner. Alternatively, thewedge-shaped microchip 120 may be positioned in a pie-shaped fashion ona platter 191 with other wedge-shaped microchips 120 a, 120 b, . . . ina rotary configuration, as shown in FIG. 11A. The platter 191 rotatesbeneath a detection assembly 202 such that portions (along line 200) ofeach wedge-shaped microchip pass by detection assembly for scanning, asshown in FIG. 11B.

[0101] Another version of the microchip 120 has a rotary geometry tosimplify loading and detection (also see FIGS. 11A and 11B). The chip isa single rotatable disk shaped structure with embedded channels 194. Thechannels 194 are radially disposed, and extend inwardly to terminate atthe same point. Samples are electrophoretically driven radially towardthe center of the disc from electrode wells 190 to electrode well 192.Samples are injected by introducing samples at wells 194 driving themtoward wells 196, followed by a switching operation. In one mode of use,the device is spun under (or over) a single point detector or thedetector is spun under (or over) the device. The detector then scans acircle of detection zones 200 near the center of the device. In thisgeometry the channels 194 are arranged in a radial pattern around adisc. The electrophoresis current is arranged to run from the peripheryof the disc toward the disc center. During sample loading, the disc isrotated in a stepwise fashion and the robotic arm with its multi-tippedsample transfer arm is brought from the sample tray to the disc asillustrated in the figures. In principle this eliminates one axis ofmotion that would otherwise be needed for the load robot. In oneembodiment of the read-out method, the disc is spun continuously duringthe electrophoresis run. A laser fluorescent or similar detector 202remains aimed at a stationary point, for example, at several millimetersradius from the chip center. The rotary rate of the disc revolution iskeyed to the gating period of the detector, so that the signal from eachchannel is synchronized with the data acquisition. This permits datafrom multiple channels to be multiplexed in time from a single detector.

[0102] Referring to FIGS. 11A and 11B, consider the case of loadingsamples from an industry-standard 96-well micropipet plate. A pipet headwith 8 tips arranged in a linear array with 9 mm spacing would be put ona robotic arm. The arm must have two linear axes of motion, X and Z, soas to permit the pipet head to pick up 8 samples and to then depositthem into 8 wells on the rotary chip, also spaced at 9 mm center. Thisarrangement is possible, for example, by using a 5 inch diameter chipand by arranging sample load ports in linear arrays with 9-mm wellspacing around the periphery of the disc. If 24 arrays of 8 wells arearranged around the 5-inch disc then a total of 192 lanes can arrangedon a single disc. The chip is loaded with a total of 24 robotic cycles.During the read-out, for example, the disc can then be spun at a rate of600 rotations per minute (10 rotations per second). The laser pointdetector will then be able to read each channel at the rate of 10readings per second.

[0103] It should be understood that the apparatus of FIGS. 1 and 4 withtest modules 32 form microelectrophoresis systems for processingbiomolecular analyte (e.g., portions of DNA such as STRs) with improvedthroughput over conventional systems. These systems provide means fororder-of-magnitude improvements in parallelism and reductions in costthrough a fully integrated process flow organized around a microchip,i.e., a unique microchannel device preferably having a verticalinjection channel. Each microchip can support a channel set lane density(i.e., the number of channels per unit area) greater than 30 lanes percentimeter. The vertical injection channel enables scaling of theinjected sample to less than 100 nanoliters, including a sample of lessthan 10 nanoliters and of less than 5 nanoliters. Analysis of simple DNAtyping can be carried out rapidly (e.g., in 30 seconds or less). Inaddition, the reduction in required sample injection permits verysubstantial (order of magnitude) reductions in the consumption ofexpensive reagents such as enzymes.

[0104] The DNA typing procedure performed using the apparatus of eitherFIG. 1 or FIG. 4 involves an allelic profiling assay for the analysis ofSTRs which can be carried out more rapidly than is possible usingconventional methods and capillary or slab gel systems. Such a proceduremakes it possible to genotype a single STR locus in 30 seconds or lessand to rapidly analyze an STR system consisting of multiple loci (e.g.,to analyze an STR system consisting of 4 loci in less than 2 minutes).

[0105] In one embodiment, the microchip is microfabricated to producechannel structures which can contain an injection plug whose width is100 μm or less. The narrow injection plugs result in short separationdevices and, therefore, shorter analysis times and reduced diffusion. Inone embodiment, the injection plug width is about 100 μm or less, suchas between 75 μm and 100 μm, between 50 μm and 100 μm, between 25 μm and75 μm, between 25 μm and 50 μm or between 25 μm and 100 μm. In aparticular embodiment, the separation device includes a microfabricatedchannel of about 45 μm×100 μm in cross section and about 20-30 mm (e.g.,26 mm) in length. The channel is filled with a replaceable matrix(injection fluid), such as a replaceable polyacrylamide matrix, which isoperated under denaturing conditions at a temperature of about 50° C.

[0106] As will now be explained, particular components of the apparatusof FIGS. 1 and 4 are available as over-the-counter parts. For example,for the apparatus 90 of FIG. 4, the load assembly 94 may use agantry-style XYZ robotic motion system operating with linear brushlessmotors in the X,Y axes, with 500×800 mm travel and 5-μm (AerotechIncorporated, Pittsburgh, Pa.). The Z axis can be controlled with a DCservo motor to 2 micron accuracy (25 mm travel). The camera 108 may beimplemented with an optical system (Edmund Scientific, Inc.) mounted ona tool platform of the load assembly 94. Motion may be programmed usinga PC (Compaq, Inc.) and driver software (Aerotech, Inc.) to control themotion of the robot and allow it to perform pre-programmed, automatedrepetitive procedures. Affixed to the tool platform of load assembly 94can be an array of eight stainless steel fluid handling tips 100(Hamilton, Inc, Reno, Nev.), fixed on 9 mm centers in order to match themicrotitre well spacing, coupled via 0.030 inch outer diameter Teflontubing to high-precision pumps (Cavro Instruments, Inc., Sunnyvale,Calif.). Each tip may be placed into a relative position with anaccuracy of plus or minus 2 mils.

[0107] In connection with the test module 32, once the microchip isloaded, the electrophoretic separation can be implemented to separatecomponents within the samples based on their molecular weight andmobility. In particular, separation through the ME device sieving mediumcan be implemented by applying a series of switched DC fields to drawanalyte down the microfabricated channel of the ME device. The biasapplied can be 100 to 800 volts per cm of channel length. An opticallaser-induced fluorescence system (Omnichrome, Inc.) can be used toexcite fluorescently tagged DNA samples (e.g, CCTV forensic diagnosticsamples, Promega Corp.) During electrophoresis the resultant signal canbe collected by a 50× microscope objective (Bausch and Lomb, Rochester,N.Y.) and photomultiplier tube (I P28, RCA Corporation) and by acharge-coupled device (Princeton Instruments, Inc., Princeton, N.J.) andanalyzed using software appropriate for the separation being done.Dichoic filters can be used to collect signal and to reject laser light,using methods known to those skilled in the art of capillaryelectrophoresis.

[0108] It should be understood that the robotic devices of the apparatusin FIGS. 1 and 4 are capable of moving the tip array of the fluidtransfer devices with complete degrees of motion (x-, y-, z-motion). Thepurpose of this arrangement is two-fold; first, to collect microliterand sub-microliter amounts of fluid sample from industry standardmicrotitre plates and subsequently position and dispense these samplesonto the microfabricated vertical injection ports of the ME device andto provide support for (or even serve as) one set of electrodes. Thisdevice structure is used in combination with a robotic fluid handlingsystem to provide ultra-high-density sample injection into the MEdevice.

[0109] Further details of the operation, preparation and manufacture ofthe vertical injection channel microchip will now be provided. Thevertical injection channel has two well-aligned holes leading to the topand bottom surfaces of the microchip, as shown in FIGS. 9A and 9B. Thesample material is put in contact with the top hole, and anelectrophoretic current is drawn from top to bottom using electrodes.Once the sample stream is well established in this vertical injectionchannel, the sample trapped at the intersection of channels and isswitched so as to move down the separation channel. This is accomplishedby switching the electrophoresis current so as to drive the samplevolume from the intersection of the channels and down the (horizontal)separation channel, i.e., from one electrode of the separation channelto another electrode of the separation channel. Preferably, the topplate is coated on its exterior surface with a hydrophobic agent. Theindentation well 164 is treated with a hydrophilic substance to greatlyalleviate the robotic positioning of sample.

[0110] To form the vertical injection channel microchip, two fusedsilica plates 180,182 micrometers thick from Hoya Corporation areprepared in the geometry of FIGS. 9A and 9B. Channel structures 150,152and indentation wells 164 are etched into the top plate 180 usingphotoresis (Shipley Corporation) to pattern an evaporated chromiumlayer. Using this chromium layer as a masking layer, 50 micrometer-deepstructure was etched into the top plate 180 with a NH₄F/HF (1:1) etchantat 50° C. A CO₂ laser system is then used to create 75 micrometervertical channels 150 through the process of laser ablation on both topand bottom plates. Following the laser ablation step, both top andbottom plates 180,182 are briefly etched in the NH₄F/HF (1:1) etchantfor 60 seconds. This leaves the surfaces of both plates in a hydrophobiccondition.

[0111] Approximately 400 microliters of sieving gel (either 2% linearpolyacrylamide of 5,000,000 molecular weight dissolved in TAPS buffer,or hydroxyethylcellulose dissolved in TAPS buffer) is placed between thetwo plates 180,182, each measuring approximately 45 cm² in area. The twoplates 180,182 is carefully aligned and compressed together. Thisprotocol succeeds in sealing the microfabricated channels andmaintaining the sieving gel intact within them. No cross-contaminationof adjacent channel sets as close as 200 μm is evident with thisprocedure.

[0112] A fluorescein-labeled PCR primer (Perkin-Elmer Corp.) Isintroduced through the indentation well 164, and the switchedelectrophoresis protocol described above is conducted at an injectionvoltage of 200 volts per centimeter of channel length. In oneembodiment, an argonion laser operating at 488-514.5 nm wavelength isused in combination with a filtered photomultiplier tube as a detectorin the manner well understood by those skilled in the art ofelectrophoresis.

[0113] The precision of the motion system is significantly relaxed usingthe concept of a hydrophobic moat. According to this concept, the bulkof the top ME device surface may be treated to form a hydrophobicsurface, and the sample loading wells 164 are coated or filled with ahydrophilic substance. As a result, fluid dispensed by the fluidtransfer system is selectively attracted to the loading wells. In oneembodiment of the hydrophobic moat concept, the ME device is brieflyetched in a hydrofluoric-acid-based etching solution to create ahydrophobic top surface. The microchip is then injected withpolyacrylamide solution (which is hydrophilic), and a small layer ofthis injected material is left in the indented wells 164. Those skilledin the art will know of alternative methods of treating ME devicestructures so as to achieve local areas of hydrophilic and hydrophobiccharacter.

[0114] In one embodiment in which the microchip is designed to achieve 3channel per millimeter channel density, channels of 50 micron width areetched at 333 micrometer spacing and terminated with sample wells of 500micron diameter in a staggered array. The accuracy of the placement ofsamples is therefore relaxed by one order of magnitude from about 50microns to about 500 microns. Relaxing the positioning accuracy and costof the motion system robotics reduces set-up time when replacing the MEdevice.

[0115] In one embodiment of the invention, electrically conductingstainless steel tips (Hamilton Corporation, Reno, Nev.) of innerdiameter 150 μm and outer diameter 720 μm set in a linear array on 4.5mm or 9 mm centers, matching the well-to-well spacing of currently usedindustry-standard microtitre plate wells. These tip arrays have beenattached to a robotic system, operated with proper software protocols,which have successfully placed them within 10 μm of the injection portson quartz ME devices, well within the tolerances of successful loadinginto the 100 μm diameter injection port size.

[0116] Further details of the fluid transfer device 42 of FIG. 2 willnow be provided. The fluid transfer device 42 is based on the well-knownsolid-phase reversible immobilization (SPRI) technique introduced byHawkins et al. (Hawkins et al., 1994) by which single-stranded DNA isbound to paramagnetic 1 μm iron oxide spheres (Bangs LaboratoriesEstaphor M0008801CN) under 13% polyethylene glycol 8000 (PEG) and 10 mMMgCl₂ salt concentrations (Paithanker and Prasad, 1991). Followingbinding of DNA to the paramagnetic spheres in an industry-standardmicrotitre plate, the assembly shown in FIG. 2 is used to transfer theDNA and spheres into the microchip, where the DNA is subsequentlywashed, eluted, and denatured in situ inside the microchip.

[0117] In FIG. 2, a nickel wire (the core 52) is embedded in anon-magnetic cylindrical piston. The piston/wire assembly is positionedin a non-magnetic housing such that motion of the cylinder in the x- andy- directions is minimal. The nickel wire is magnetized by applying acurrent to solenoid coils (the winding 54) which surround a portion ofthe nickel wire. Once magnetized, the iron oxide beads coated with thessDNA adhere magnetically to the exposed nickel wire tip 58. The fluidtransfer device 42 is then precisely positioned over the injection portof the microchip of test module 32, and the exposed nickel wire tip isinserted into this entrance port. A compressed spring and hard stopallow retraction of the tip in case of unintended or unsuccessfulplacement without damage to the ME device or wire tip. An electromagnetpositioned underneath the microchip is then energized while the solenoidin the assembly is de-energized, and the ssDNA-coated beads are thusheld inside the injection port vicinity inside the NM device. Whilemagnetically held in place, the ssDNA beads are then subsequentlywashed, eluted, and denatured. The released DNA is then electrophoresedand separated in the separation channel of the ME device, while the nowuncoated magnetic beads are held in the injection port area by theelectromagnet. Methods of denaturing the ssDNA after they have beenreleased from the beads include increasing the temperature of the MEdevice with an external heating unit, or pulsing the current through theelectromagnet to induce local heating in the vicinity of the injectionport. Initial experiments with such a nickel wire system coupled to asolenoid successfully demonstrated collection of paramagnetic μm ironoxide spheres (Bangs Laboratories Estaphor M0008801CN); the solenoid tedof approximately 500 turns of copper wire carrying 200 mA with an innerair core diameter of 5 mm, and a 0.005 inch diameter piece of wireapproximately 1 cm in length.

[0118] The present invention is further illustrated by the followingexamples, which are not intended to be limiting in any way. The examplesare similarly described in an article entitled “DNA typing in thirtyseconds with a microfabrication device”, by Schmalzing et al., Proc.Natl. Acad. Sci. USA, Vol. 94, pp. 10273-10278, September 1997,Genetics, the teachings of which are hereby incorporated by reference intheir entirety.

EXAMPLES

[0119] Methods and Materials

[0120] The following methods and materials were used in the examplesdescribed herein.

[0121] Micromachining.

[0122] Miniaturized electrophoresis devices were fabricated usingphotolithography and chemical etching methods to produce channelstructures in fused silica wafers. A 40-nm-thick film of chromium wassputtered onto 150-mm-diameter 0.4-mm-thick fused silica wafers (Hoya,Tokyo, Japan). 1811 Photoresist (Shipley, Marlborough, Mass.) wasspin-coated onto the wafer to a thickness of 1.1 mm and baked at 90° C.for 25 minutes. The resist was patterned by selective exposure to 365-nmUV light (UVP, Upland, Calif.) through a contact photomask withlinewidths of 10 μm (Advanced Reproductions, Wilmington, Mass.) anddeveloped with Microposit photoresist developer (Shipley). Theselectively exposed chrome was removed using K₃Fe(CN)₆/NaOH chrome etch(Shipley). The resulting mask pattern was etched into the fused silicaby immersing the wafer in NH₄F/HF (1:1) etchant at 50° C. The depth ofetching was controlled by monitoring etching time and measured with aprofilometer. Photoresist was removed with acetone, and the remainingchrome was dissolved using K₃Fe(CN)₆/NaOH. Access to the channel endswas provided by 75-mm-diameter holes drilled through the etched waferwith a CO₂ laser system. A second 150-mm-diameter fused silica wafer wascontact bonded to the etched wafer to enclose the channels. To achievebonding, both wafers were immersed in a bath of NH₄OH/H₂O/H₂O₂ 4:5:1 at50° C. and then rinsed thoroughly with filtered water. They were thenplaced in direct contact and thermally bonded. Initial reversiblebonding took place at 200° C. (2 h), followed by final permanent bondformation at 1000° C. overnight. This is well below the softening pointof the fused silica substrate utilized and, therefore, the bonding isdue to the formation of covalent bonds between the two surfaces and notdiffusion of boundary molecules. Individual microchips were cut from thebonded wafer pair using a wafer saw. Reservoirs of 50 microliters volumewere formed by affixing 5-mm-tall 3-mm-i.d. glass raschig rings (AceGlass, Vineland N.J.) with optical cement (Norland Optical, NewBrunswick, N.J.) around each exit hole.

[0123] Reference is made to a cross-structure device with a straightseparation channel in FIG. 8A. Portions of the injection channel 150 andthe separation channel 152 are hereinafter referred to as channels A, B,C and D and labeled accordingly in FIG. 8A.

[0124] Channels A, B and C are 5 mm long and the separation channel Dhas a length of 30 mm. The chip was isotropically etched to a depth of45 μm, producing a channel with a semi-circular cross section and awidth of 100 μm at the top. The cross sectional area of the channels isequivalent to that of a cylindrical capillary with an internal diameterof 70 μm. Other structures were fabricated to study the effect ofchannel folding, a common technique used to create compact structures inmicrofabricated devices. In these other structures, channels A, B and Care 5 mm long and the folded separation channels are 100 and 300 mm inlength. The etch depth for the folded devices are 60 μm, resulting in130 μm-wide channels. Two different intersection geometries werefabricated in the folded channel chips. In the first case all fourchannels meet at a single point, while in the other, channels A and Care offset vertically by 250 μm.

[0125] Coating.

[0126] The inner channel surfaces of these microfabricated devices werecoated using a modified Hjerten procedure. A filtered solution of 1.0 MNaOH was flushed through the channels for 10 min followed by a 12-houretching period. The channels were subsequently rinsed with filtereddeionized water, 0.1 M HCl, deionized water, and MeOH, 10 min each, andthen dried in a stream of He 6.0 (Boc Group, Murray Hill, N.J.). Thechannels were then rinsed for 10 min with a filtered solution consistingof 5.0 mL 95% MeOH/water, 0.5 mL 10% HAc and 1.0 mL3-(Trimethoxysilyl)propylmethacrylate (Fluka, Buchs, Switzerland). After12 hours, the channels were rinsed first with MeOH then with deionizedwater, 10 min each. The channels were again dried with He 6.0. In thelast step, 70 mg of acrylamide (Pharmacia, Uppsala, Sweden) dissolved in1.0 ml of separation buffer (see below) and kept air-tight in a glassvial with septum was purged with He 6.0 at room temperature to removeany traces of oxygen. After 2 hours, 8 μL each of 100% TEMED (Sigma, St.Louis, Mo.) and of 20% APS (Pharmacia, Uppsala, Sweden) in water wereadded by a syringe to the acrylamide solution. After brief vortexing,the mixture was pulled up through the septum into a syringe and pressedinto the etched channels. After 12 hours of polymerization at roomtemperature excess polymerized coating solution was purged from thechannels using a syringe. The channels were then ready to be filled withthe replaceable separation matrix.

[0127] Separation Matrix.

[0128] The working buffer consisted of 1×TBE with 3.5 M urea and 30% v/vformamide (Pharmacia, Uppsala, Sweden). A solution of 4% acrylamide w/vin working buffer kept in a glass vial equipped with a septum, waspurged with He 6.0. After 2 hours two mL each of 10% TEMED and 10% APS(both in water) were added with a syringe through the septum. Themixture was briefly vortexed and allowed to polymerize for 12 hours.Aliquots of the matrix (stored at 4° C.) were transferred into a syringeand pushed into the coated microchip channels. This operation wasperformed with the aid of a mechanical fixture and could be fullyautomated in future generations of the apparatus.

[0129] Robotic Sample Preparation.

[0130] A robotic system was used to prepare the STR samples. Thisconsisted of T265 robotic arm on a linear track (CRS Robotics,Burlington, OT, Canada), a microplate feeding station (Eastern TechnicalSales, Manchester, N.H.), a liquid pipetting station (Rosys, Wilmington,Del.), a heat block, and Progene thermal cyclers (Techne, Princeton,N.J.). Bloodstain card punches in 96-well Cycleplates (RobbinsScientific, Sunnyvale, Calif.) were washed with FTA Purification Reagent(Fitzco, Minneapolis, Minn.), TE (1 mM Tris-HCL, 0.5 mM EDTA, pH 8.0),and ethanol. A volume of 30 mL of PCR mix [1×STR buffer (Promega,Madison, Wis.), 5′-fluorescein labeled CTTv Quadruplex primer pairs (5mM of each primer) (Promega), bovine serum albumin (60 mg/mL) andAmplitaq Gold DNA polymerase (50 mg/mL) (Applied BiosystemsDivision-Perkin Elmer, Foster City, Calif.)] was added to each well,followed by 25 mL of liquid wax (MJ Research, Watertown, Mass.). Thermalcycling was attained at 95° C. for 10 min, 10 cycles of 94° C. for 1min, 60° C. for 1 min, and 70° C. for 1.5 min, 20 cycles of 90° C. for 1min, 60° C. for 1 min, and 70° C. for 1 min, and 70° C. for 10 minutes.

[0131] Instrumentation.

[0132] A schematic of the microchip genotyping apparatus is shown inFIG. 7. In brief, a fused silica microchip was mounted on atemperature-controlled stage with high voltage connections and opticalaccess for laser induced fluorescence (LIF) detection. The microchip wasaffixed to an Al₂O₃ alumina heater block whose temperature wascontrolled by a temperature controller (Omega Instruments,Stamford,Conn.) and a series of thermocouples. The alumina block contained amachined aperture to allow for optical access of the detection zone.

[0133] High voltage was provided to platinum wire electrodes mounted inthe four glass fluid reservoirs by a SL150 power supply (Spellman,Plainview, N.Y.). The voltages applied to each reservoir were controlledby a manual switching circuit and a resistor-based voltage dividernetwork. Laser-induced fluorescence detection was achieved using anInnova 90 argon ion laser (Coherent, Santa Clara, Calif.) operating alllines. The beam was focused to a spot size of 15 μm in the channel at30° C. angle of incidence with a 10 cm focal length lens. A 50×, 0.45N.A. long working distance microscope objective (Bausch & Lomb,Rochester, N.Y.) collected the fluorescence emission. The collectedlight was spatially filtered by a 4-mm-diameter aperture in the imageplane and optically filtered by two 520DF20 bandpass filters (OmegaOptical, Brattleboro, Vt.) and detected by a photomultiplier detectionsystem. The PMT current signal was converted to voltage across a 100k_resistor, digitized with a PC-controlled bit data acquisition system(Data Translation, Marlborough, Mass.), and analyzed using C Gramssoftware (Galactic Industries, Salem, N.H.).

[0134] Microchip Separations.

[0135] For fast genotyping, all four channels of the chip described inFIG. 8A were filled with the polyacrylamide separation matrix through asyringe interfaced to the separation channel exit hole. The detector wasplaced 26 mm from the injector. The freshly filled chip waspre-electrophoresed for 3 min at 200 V/cm across the separation channelat 50° C. To separate the CTTv internal standard ladder, 2 μL of theladder was diluted with 8 μL working buffer. For the allelic profiling,4 μL of PCR amplified sample was added to 2 μL of CTTv ladder anddiluted to a total volume of 10 μL with 2×TBE buffer (3.5 M urea, 30%v/v formamide). The samples were briefly vortexed, denatured for 2 minat 95° C., chilled on ice and pipetted into the microchip sample viallocated at the end of channel A. The chip was operated in the pinchedcross injection mode, in which three ionic currents are merged at theinjection point to confine the sample ions to the volume defined by thechannel intersection. To load the sample, 400 V/cm was applied acrosschannels A and C. Field strengths of 40 V/cm were applied to channels Band D to prevent the sample from entering these channels. This resultedin a stable injection plug length of 100 μm and an injection volume ofapproximately 0.36 nL. To inject the representative sample plug into theseparation channel, the voltages were switched to create a fieldstrength of 200 V/cm in the separation channel, and approximately 20V/cm in the channels A and C. This generated a well defined plugentering the separation channel D with no excess leakage of sample fromthe side channels A and C. For experiments at higher field strengths,all voltages were equally multiplied. Similar field strengths were usedin the 100 mm and 300 mm folded channel microchip devices resulting ininjection plug lengths of 130 μm and 250 μm respectively, depending onthe intersection geometry. Samples were changed by rinsing the samplereservoir A three times with separation buffer, loading the new sample,and pre-electrophoresis for several seconds through the cross channel toprevent sample carry-over.

[0136] Slab Gel Electrophoresis.

[0137] Three microliters of 5′-fluorescein labeled CTTv ladder (diluted1:5) was mixed with 3 μL of formamide containing 5′-Rox labeledGenescan-2500 size standard (Applied Biosystems Division Perkin Elmer,Foster City, Calif.). The sample was maintained at 95° C. for 2 min,quickly cooled in ice, and electrophoresed for 2.5 hours at 28 W througha denaturing 8% polyacrylamide gel in Applied Biosystems 373 DNAsequencer running Genescan software. The sizes of the CTTv ladder andPCR products were automatically determined by Genescan analysis softwareusing the local Southern method. The polyacrylamide gel was pre-run for20 min before loading the samples.

Example 1 Determination of Factors which Influence the Speed of STRAnalysis

[0138] Initially, the general operation of the microfabricated devicefor genotyping by STR analysis was characterized. The objective was todetermine the factors which influence the ultimate speed of suchanalyzes in microchip devices. Device performance was followed for theSTR system CTTv, which consists of the four loci CSF1PO, TPOX, THO1 andvWA, each of which contains STR alleles which differ in length by fourbase pairs. The four loci, CSF1PO, TPOX, THO1 and vWA, contain 9, 5, 7,and 8 common alleles respectively. FIG. 12A shows the separation of theCTTv ladder ranging from 140 to 330 bases by microchip gelelectrophoresis using one of our devices. This ladder was used as aninternal sizing standard for the allelic profiling. In FIG. 12A thealleles of all the four loci are well resolved in less than two minuteswith measured resolution R,

R=([2ln2]^({fraction (1/2)})() t ₂ −t ₁)/(hw ₁ +hw ₂)  (1)

[0139] where t_(n) is the retention time of the nth peak and hw_(n) isthe full width at half-maximum of the n^(th) peak. The resolution rangesfrom 1.7 for the vWA locus to 1.1 for the CSF1PO locus. We chose aminimum resolution of R=1.0, which is typically required for forensicapplications, as a requirement while we optimized for analysis speed.This level of resolution is easily achieved in our microchip in aseparation time that is approximately two orders of magnitude fasterthan conventional slab gel electrophoresis.

[0140] The interdependent parameters varied were operating temperature,channel shape (straight or serpentine), field strength, injection pluglength, and channel length. Injection plug length was varied from 100 μmto 250 μm using simple cross and offset cross injectors. Channel lengthwas varied between 13 mm and 295 mm.

[0141] After initial experiments at room temperature, the device wasoperated at 5° C. throughout the optimization process. Heating of thematrix assisted in keeping the samples denatured, and resulted in anearly two-fold decrease in analysis time when compared to ambienttemperature, this decrease is attributed to a decrease in the viscosityof the sieving gel. There were no observed changes in selectivity orpeak width relative to operation at room temperature. Temperatures above50° C. were found to be impractical, primarily due to bubble formationin the channels.

[0142] For STR analysis, serpentine channel bends were found tosignificantly degrade device performance. A band-broadening effect wasobserved which can be quantitatively explained by a simple geometricalcalculation of the path length differences introduced by the turns,under the assumption that cross-channel migration randomizes molecularpaths completely between turns. In contrast, analysis of data fromstraight channel devices revealed that these separations wereessentially injection-plug limited. The absence of other measurable bandbroadening effects underlines the near ideal performance of ourmicrochip gel system.

[0143] The field strength determines the migration speed within thedevice and influences the performance of the sieving matrix.Experimentally, the field strength was increased from values typical forcapillary gel electrophoresis (approximately 200 V/cm) to values as highas 800 V/cm. At high fields, the resolution suffered due to the onset ofnew molecular sieving mechanisms such as biased reptation. The highestfield at which a resolution of R=1 was maintained depended on thespecific locus. As an example, FIG. 13 displays results of a separationat 500 V/cm where the vWA locus is baseline separated in thirty seconds.At a field strength of 800 V/cm the device performance becameunpredictable and required replacement of the polyacrylamide solution torecover performance. Below 600 V/cm the device and sieving matrixexhibited excellent long term stability. An increase in migration timesof about 10% was found during the course of 10 consecutive runs.However, the original migration times could be restored by replacing thegel-buffer system. In addition, the accuracy of the allele assignmentwas not affected by small changes in migration time since an internalstandard was used for allele identification. No other changes inseparation were observed even after 20 consecutive runs withoutreplacement of the gel-buffer system. Occasionally a high fluorescencebackground was seen which was most likely due to contamination of theinjection or detection zones with dust particles. Replacement of thegel-buffer system consistently restored the background signal to normal.A single microchip device was used for allelic profiling for an entireweek (about 20 to 30 separations per day) at 50° C. with no noticeabledeterioration. The polyacrylamide matrix was routinely replaced everymorning after it was found that overnight storage of the chip at 4° C.could lead to a decrease in performance. The wall coating showed nodegradation during continuous use over the course of one week.

[0144] The channel length required for a minimum resolution of R=1depended primarily on field strength and injector length. The differentinjectors were characterized by varying field strength and effectivechannel length (the latter by moving the position of the detector alongthe length of the microfabricated channel). Minimum channel length witha given injector and given field is set by the acceptable resolution forthe locus of interest.

Example 2 Assay of PCR Amplified Samples

[0145] The microchip device was used for genotyping of PCR amplifiedsamples of eight individuals which were spiked with the CTTv ladder asthe internal size standard and assayed on the microchip gel system. Inall eight cases, the alleles could be identified with no ambiguity inunder two minutes and the results were in complete agreement with dataproduced by traditional slab gel electrophoresis, which typicallyrequired 80, 94, 112 and 143 min to detect and resolve the alleles ofvWA, THO1, TPOX, and CSF1PO respectively. The spiking experiment for oneof the individuals is shown in FIG. 12B. The individual is clearlyhomozygous for vWA (allele 14) and heterozygous for THO1 (alleles 7/9),TPOX (alleles 8/9) and CSF1PO (alleles 10/14).

[0146] Results, thus, have demonstrated that the quadrupled STR systemCTTv can be analyzed with high accuracy in less than two minutes and asingle locus in 30 seconds by microchip gel electrophoresis. Compared tocapillary or slab gel electrophoresis, the approach described hereindevice is faster by a factor of ten or one-hundred, respectively. Inaddition, the present system can be operated for an extended period oftime in a highly reliable manner.

[0147] The high speed of analysis can be explained by the fact thatmicrochips allow very short and precisely controlled injection plugwidths (100 μm and less). These narrow injections permit shortseparation distances and consequently shorter analysis times and resultin reduced diffusion. The influence of a given injection plug length ontotal analysis time can be estimated according to Eq. 2

R _(t)=0.25DμtE(s ₁ ²+2Dt)^({fraction (−1/2)})  (2)

[0148] where the theoretical resolution R_(t) is calculated under theassumption that injection and diffusion are the sole contributors topeak width. In this expression, Dμ is the difference of mobility of twoneighboring DNA fragments, E is the electric field strength, t is thefragment migration time, s₁ ² is the variance due to injectionbroadening, and 2Dt is the variance of the diffusion contribution withthe diffusion coefficient D. Other effects, such as broadening due todetection or thermal gradients across the channel were not observed inour system. Optimal performance was only obtained when straightseparation channels were used, since in folded channels, as discussedpreviously, the broadening effect of the channel bends dominated.

[0149]FIG. 14 shows the calculated separation times required to achievea resolution of R_(t)=1 for the last pair of alleles in the CSF1P0 locusas a function of injection width at two different field strengths. Sincethis locus is always the most difficult to separate it determines theoverall CTTv analysis time for the full quadruplex system. Thesecalculations are based on electrophoretic mobilities and diffusioncoefficients determined experimentally for the CTTv ladder in ourmicrochip gel system. Two extremes can be distinguished for theseparation at 200 V/cm. In the case where the injection plug is largerthan 100 mm, the minimum separation time is linearly dependent on plugwidth and independent of diffusion. Below 10 mm injection width thesituation is reversed; the system becomes diffusion limited and almostindependent of injection width. Any further decrease in injection pluglength will therefore not result in a significant decrease in minimumseparation time. The theoretical time in the limit of zero injectionwidth is predicted by Eq. (2) with s_(I)=0. In this limit, resolution isdetermined by parameters which are solely gel dependent. A minimumseparation time of 26 seconds for the CTTv system, corresponding to aseparation distance of 6 mm, was calculated for 200 V/cm. This is only afactor of 4 faster than what was obtained in this study where we havebeen restricted by available devices to a minimum injection width of 100μm. The predicted result would require a 10 μm injector, which in oursystem would not be feasible without an improved detector, or apre-concentration step of the STR samples. However, we consider aninjection plug length between 25 μm and 50 μm as being practical forroutine STR analysis with our system. The curve for the separations at500 V/cm shows a similar behavior with even greater improvementpredicted for scaled down injectors. A device utilizing injectorsbetween 25 μm and 50 μm with a separation length of 14 mm at 500 V/cmshould result in a total separation time of 15 to 25 seconds for thefull CTTv ladder. Despite the observed loss in selectivity at 500 V/cmprimarily due to biased reptation, adequate resolution is maintainedbecause of the decreased diffusional broadening due to short analysistimes with the higher field.

[0150]FIG. 15 shows the same analysis at a field strength of 500 V/cmfor each of the four loci of the CTTv analysis. As above, thecalculations are based on electrophoretic mobilities and diffusioncoefficients measured for the CTTv ladder in our microchip gel system.The first three loci show very similar behavior. Extremely high speedanalysis should be possible for these lower molecular weight fragments.It should be possible to perform STR analysis of the first three lociusing a 25 μm injector in less than 4 seconds.

[0151] Thus genotyping for a single locus has been performed in 30seconds and the CTTv STR system consisting of four loci has beenanalyzed reliably in less than two minutes in a microchip system. Thedevice is already highly optimized and can perform repeated analyseswithout replacement of the sieving matrix. Further optimization willoccur with an improved fabrication geometry which would allow injectionplugs between 25 μm and 50 μm in length. The current device uses samplevolumes of 4 μL from standard PCR preparations, although the use of evensmaller volumes should be possible without loss in performance. Thecurrent microchip system offers an improvement in speed over currenttechnology of almost two orders of magnitude, with no compromise inquality for standard quadruplex CTTv analyses.

Example 3 Evaluation of the Feasibility of Loading Schemes

[0152] A glass microchip with laser-drilled injection ports leading tomicrochannels was interfaced with a 1 cm thick gasket fabricated frompolydimethylsiloxane (PDMS) silicone elastomer material (Dow ComingSylgard 184, Schenectady, N.Y.). Capillaries of inner diameter 100 μmand outer diameter 375 μm were molded into the gasket and set in placewhile the PDMS cured to a semi-rigid final state. No rigid supportstructure was used in these trials, and the capillary-gasket system wasaligned to the injection holes on the microchip manually with the aid ofa microscope. The free end of the 10 cm long capillary was inserted intoa container of fluorescein and a voltage of 50 volts per centimeter wasapplied between the container and the microchip channels. The microchipwas illuminated with an argon ion laser source operating at 488 nm, andthe fluorescing fluorescein was observed flowing from its originalcontainer to the microchannels via the elastomer gasket with no leakageat the interface between the gasket and the quartz microchip.

[0153] The elastomer gasket, with the capillary molded into it, wassubsequently removed from the microchip, cleaned by rinsing with astream of water, dried, and then replaced on the microchip. Theexperiment was repeated successfully over ten times, after which theinterfacial surface of the elastomer gasket became sufficientlycontaminated with dirt, primarily due to handling, that a hermetic sealwas no longer able to be achieved.

[0154] While this invention has been particularly shown and describedwith references to preferred embodiments thereof, it will be understoodby those skilled in the art that various changes in form and details maybe made therein without departing from the scope of the inventionencompassed by the appended claims.

What is claimed is:
 1. A method for loading a test plate with a sampleof biomolecular analyte that is disposed about a bead that ismagnetically attractable, the method comprising the steps of: activatingan electromagnetic loading device to electromagnetically attract andcarry the bead; positioning the electromagnetic loading device adjacentan opening of the test plate; and deactivating the electromagneticloading device to release the bead at the opening of the test plate suchthat the sample of biomolecular analyte is loaded on the test plate. 2.The method of claim 1 further comprising the step of: activating anelectromagnetic unloading device that provides a force on the bead in adirection away from the electromagnetic loading device and toward theopening of the test plate.
 3. The method of claim 1 wherein the step ofpositioning the electromagnetic loading device includes the step of:actuating a robotic assembly that supports the electromagnetic loadingdevice such that the electromagnetic loading device moves to aprogrammed position relative to the test plate.
 4. A method for loadinga test plate with a sample of biomolecular analyte that is disposedwithin a solution, comprising the steps of: positioning a capillaryadjacent an opening of the test plate; activating an electrokineticsdevice coupled to a capillary such that the sample is drawn through thecapillary onto the test plate using electrokinetics; and deactivatingthe electrokinetics device such that the sample is no longer drawnthrough the capillary.
 5. The method of claim 4 wherein the step ofactivating includes the step of: dispensing a fluid droplet of thesample onto the test plate.
 6. A test module assembly, comprising: adielectric plate member having an upper planar surface and a lowerplanar surface that is spaced apart from and coplanar with the upperplanar surface, the dielectric plate member having at least one set ofchannels that includes an injection channel and a separation channel,the injection channel extending from the upper planar surface to thelower planar surface, the separation channel extending within thedielectric plate member in a plane parallel with the upper and lowerplanar surfaces and intersecting the injection channel.
 7. The testmodule assembly of claim 6 wherein the injection channel intersects theseparation channel at a right angle.
 8. The test module assembly ofclaim 7 wherein the injection channel terminates at the upper and lowerplanar surfaces at right angles to the upper and lower planar surfaces.9. The test module assembly of claim 6 wherein the injection channelterminates at the upper and lower planar surfaces at right angles to theupper and lower planar surfaces.
 10. The test module assembly of claim 6wherein the dielectric plate member is wedge shaped.
 11. The test moduleassembly of claim 6 further comprising: a housing that attaches to thedielectric plate member, the housing providing (i) a first opening overthe upper planar surface such that a first end of the injection channelis accessible through the first opening, and (ii) a second opening overthe lower planar surface such that a second end of the injection channelis accessible through the second opening.
 12. The test module assemblyof claim 11 wherein the housing further provides a third opening overthe upper planar surface such that the separation channel is accessibleto a detection device through the third opening.
 13. The test moduleassembly of claim 11 further comprising: a heating device, supported bythe housing, that provides heat to the dielectric plate member whenpower is provided to the heating device.
 14. The test module assembly ofclaim 11 further comprising: a first porous membrane that covers thefirst end of the injection channel and a second porous membrane thatcovers the second end of the injection channel to retain a matrix withinthe injection and separation channels, the first and second porousmembranes being adhered to the dielectric plate member.
 15. The testmodule assembly of claim 11 further comprising: an electromagneticunloading device, coupled to the housing, that draws magnetic materialtowards the dielectric plate member when the electromagnetic unloadingdevice is activated.
 16. The test module assembly of claim 6 wherein thedielectric plate member includes multiple sets of channels, each set ofchannels including an injection channel and a separation channel, theinjection channel of each set extending from the upper planar surface tothe lower planar surface, the separation channel of each set extendingwithin the dielectric plate member in a plane parallel with the upperand lower planar surfaces and intersecting the injection channel of thatset.
 17. The test module assembly of claim 16 wherein the injectionchannels of the multiple sets of channels are parallel with each other.18. A method for separating a sample of biomolecular analyte, comprisingthe steps of: drawing the sample along a longitudinal axis of aninjection channel of a test plate using electrokinetics, the test platebeing planar in shape along an orthogonal axis to the longitudinal axis;and subsequently drawing the sample along the orthogonal axis through aseparation channel of the test plate using electrokinetics, theseparation channel intersecting the injection channel within the testplate.
 19. The method of claim 18 further comprising the steps of:drawing multiple samples along the longitudinal axes of multiple otherinjection channels using electrokinetics simultaneously with the step ofdrawing the sample; and drawing the multiple samples along otherorthogonal axes through multiple other separation channels usingelectrokinetics, the multiple other separation channels respectivelyintersecting the multiple other injection channels.
 20. The method ofclaim 19 further comprising the step of: scanning a respective portionof the separation channel and the multiple other separation channelswith a detection device.
 21. A test module assembly, comprising: arotatable dielectric plate member having an upper planar surface and alower planar surface that is spaced apart from and coplanar with theupper planar surface, the rotatable dielectric plate member havingmultiple separation channels that extend within the rotatable dielectricplate member in a plane parallel with the upper and lower planarsurfaces, the multiple separation channels extending radially from aninner portion of the rotatable dielectric plate member to an outerportion of the rotatable dielectric plate member.
 22. The test moduleassembly of claim 21 wherein the rotatable dielectric plate member isdisk shaped and further includes multiple injection channels, each ofthe multiple injection channels intersecting a corresponding one of themultiple separation channels within the outer portion of the rotatabledielectric plate member.